We have accumulated evidence pointing to a distinctive role of the un(der)phosphorylated form of the Rb protein during S phase that is blocked by SV40 small t Ag. Specifically, Rb can suppress the rereplication of DNA segments after DNA damage, at least in part by homing to and forming complexes with multiple genomic replication initiation elements. In preparation for post-damage replication site localization, Rb must also localize in the chromatin fraction, a process that we have shown is pp2A-dependent. Thus, small t blocks Rb/chromatin localization and, thereby, its ability to home to replication initiation sites after DNA damage. One end product of small t action in this regard was endoreduplicafion of cellular DNA. In addition, we have found that the product of the Nijmegen Break Syndrome gene, Nbs1, is a heretofore unappreciated large TAg binding protein; that Nbs1, like Rb, participates in rereplication suppression-both of cellular replicons and the viral genome; and that TAg can, upon Nbs1 binding, override this activity. The latter effect may well contribute to the established ability of T in inducing genomic rereplication as well as fully autonomous replication of circular DNA structures that contain an SV40 replication origin. In this application, we propose to search for: 1) biochemical evidence that sheds light on how Rb and Nbs1 suppress genomic rereplication; 2) for insights into where during the cell cycle the pp2A Rb/chromatin entry process is applicable; 3) for information on how pp2A operates biochemically in this process, and 4) for evidence indicating whether or not Rb and/or other pocket proteins serve as key small t targets during the performance of its neoplastic co-transforming function in human cells.